Additionally, skin irritation and histopathology studies indicated the safe and non-irritant behaviour of the NTZ-TEG. The obtained findings suggested the enhanced skin permeation and improved anti-leishmanial effect of NTZ when administered as NTZ-TEG.Topical delivery is preferable over systemic delivery for cutaneous leishmaniasis, because of its easy administration, reduced systemic adverse effects and low cost. Nitazoxanide (NTZ) has broad-spectrum activity against various parasites and has the potential to avoid drug resistance developed by enzymatic mutations. NTZ oral formulation is associated with severe dyspepsia and stomach pain. Herein, NTZ-transethosomes (NTZ-TES) were prepared and loaded into chitosan gel (NTZ-TEG) for topical delivery. NTZ-TES were prepared by the thin-film hydration method and optimized statistically via the Box-Behnken method. The optimized formulation indicated excellent particle size (176 nm), polydispersity index (0.093), zeta potential (−26.4 mV) and entrapment efficiency (86%). The transmission electron microscopy analysis showed spherical-sized particles and Fourier-transform infrared spectroscopy analysis indicated no interaction among the excipients. Similarly, NTZ-TEG showed optimal pH, desirable viscosity and good spreadability. NTZ-TES and NTZ-TEG showed prolonged release behaviour and higher skin penetration and deposition in the epidermal/dermal layer of skin in comparison with the NTZ-dispersion. Moreover, NTZ-TES showed higher percentage inhibition, lower half-maximal inhibitory concentration (IC NTZ shoLeishmania as compared to the other second-line anti-parasitic agents being more specific for bacterial or fungal infections [in vivo results indicated that by giving NTZ orally at the dose of 400 mg kg−1 d−1, the liver and splenic leishmanial parasitic load was reduced by more than 90% [−1 d−1 intraperitoneally, indicated 82% and 50% reduction in liver and splenic parasitic load, respectively [in vitro activity of NTZ has been reported against Leishmania mexicana, Plasmodium berghei, Trypanosoma curzi, Entamoeba histolytica and Trichomonas vaginalis [in vivo therapeutic activity of NTZ in BALB/c mice against Leishmania donovani was first reported by Zhang et al. [Leishmania infantum, causing visceral leishmaniasis, which showed good in vivo efficacy at low doses, thus have potential as a new anti-leishmanial agent [NTZ has potential to avoid the drug resistance developed by enzymatic mutations by inhibiting first step, i.e. causing the pyruvate binding to the cofactor thiamine pyrophosphate in opposition to the binding with potential substrates in the cell . Moreovefections . In a prthan 90% . Yet, ththan 90% . In anotectively . The in aginalis ,21, whilg et al. . Recentlal agent . In our Although topical drug delivery system has various advantages the conventional systems i.e. creams, ointments and gels cannot penetrate into the deeper skin layers ,23. Thus. 2. 2.1−1) were obtained from Thermo Fisher Scientific, USA. All other reagents used were of pure analytical grade.NTZ was purchased from Macklin Biochemical Co., Ltd . Phospholipon 90 G (PL90G) was a generous gift donated by Lipoid AG, Switzerland. Roswell Park memorial institute (RPMI-1640) medium, Medium-199 (M-199), fetal bovine serum (FBS) and penicillin/streptomycin  chitosan gel. For 3 ml preparation of NTZ-TEG, 100 mg of low- or medium-molecular weight chitosan was dissolved in 1% (v/v) acetic acid solution with continuous stirring and then 5 ml final volume was made up by adding 2 ml of PBS 7.4 having optimized NTZ-TES pellets dispersed in it. The mixture was stirred continuously until the formation of a homogeneous gel .. 2.2.3esign Expert® . The independent variables selected were lipid, surfactant and ethanol while dependent variables were particle size (PS), polydispersity index (PDI) and zeta potential (ZP) [For the optimization of blank TES, a two-level three-factors, Box-Behnken Design was used by employing the software Dial (ZP) .. 2.3. 2.3.1The dynamic light scattering method was employed to characterize NTZ-TES in terms of PS, PDI and ZP using the Zetasizer . Each measurement was recorded in triplicate (mean ± s.d.). The scanning was performed at 25°C and 90° scattering angle. ZP was determined by the use of universal-dip-cell in the Zetasizer ZS 90 ,34.The superficial morphology of NTZ-TES was examined by using transmission electron microscopy (TEM). In this analysis, the prepared NTZ-TES formulation was placed on a grid of copper coated with carbon in droplet form and allowed to stand for some time so that it got stuck to the grid. After that a 1% phosphotungstic acid was used as the negative stain and TEM analysis was then performed at 100 kV –36.. 2.3.2The indirect method was employed to determine the entrapment efficiency (%EE) of TES. The prepared NTZ-TES were subjected to centrifugation at 15 000 rpm for the period of 2 h. After the centrifugation, the supernatant was separated from the pellets, diluted with acetonitrile and then UV-visible spectrophotometric analysis was done to measure free NTZ. The following equation was then employed for the calculation of %EE ,34):% EE. 2.3.3−1 using an FTIR spectrophotometer [In order to determine the compatibility of drug and excipients, Fourier-transform infrared spectroscopy (FTIR) analysis of the pure NTZ, physical mixture (PM), PL90G, and optimized NTZ-TES was performed. About 10 mg of sample was compressed along with 200 mg of KBr into a disc using a hydraulic press and FTIR spectra was measured over the range of 500–4000 cmotometer ,38.. 2.3.4The colour, appearance and homogeneity of both the blank and NTZ-TEG was evaluated visually . A digit. 2.3.5For measurement of gel spreadability, an area of 2 cm was marked on the glass slide and gel of 500 mg was placed. Then, one more glass slide along with a 500 g weight was placed on first glass slide having gel on it for 5 min at a room temperature to allow proper spreading of the gel. After that, the increase in area of gel spread was measured and the percentage spreadability of gel was calculated by the following equation :% spread. 2.3.6in vitro study was performed for NTZ-dispersion, NTZ-TES and NTZ-TEG by using the PBS of pH 5.5 as the release medium. pH 5.5 depicts the pH of the macrophage endosomes and also the pH of skin [For investigation of the NTZ release behaviour, an  of skin . The NTZ of skin :Qn=Vr∗Cn. 2.3.7ex vivo permeation study, albino Wister rats (weight 100–125 g) were purchased from the Central Animal House of NIH, Islamabad, and were sacrificed. Their skin was isolated and used in this study. Moreover, the ex vivo permeation study was executed using a nine stations Franz diffusion cell apparatus . The rat skin with 400 µm ± 20 µm thickness was excised and clipped between the donor and receiver compartment with upwards facing the stratum corneum (SC). In the receiver compartment, the PBS (pH 7.4) was filled and stirring at 300 rpm was kept fixed. The NTZ-dispersion, NTZ-TES and NTZ-TEG were then placed in the donor compartment and a 24 h study was performed. The temperature of 32 ± 1°C was maintained throughout the experiment to simulate the skin conditions. The samples were then withdrawn at predetermined times  and analysed for NTZ using a UV spectrophotometer [For the otometer ,47.The steady state flux (Jss), the enhancement ratio (ER) and permeability coefficient (Kp) were determined by employing following equations :Jss=amouAfter completion of the 24 h study, the samples of skin were removed carefully, washed with PBS and dried by blotting to remove excess formulation. The skin was chopped into small fragments, homogenized and then sonicated in 20 ml acetonitrile for 1 h to extract the drug completely. After that the sample was centrifuged at 10 000 rpm for 15 min, and the supernatant was taken to estimate the amount of drug deposited in SC and dermal layers by using a UV-spectrophotometer .. 2.3.8−1 [Any change in the lipid organization of the epidermal skin layer produced by the permeation enhancers after treatment with the NTZ-dispersion and NTZ-TEG was evaluated by FTIR . The epi−1 ,52.. 2.3.9et al.[n = 4) and skin was shaved. One group was treated with NTZ-TEG, a second group with 0.8% formalin and a third group was kept untreated. The skin of the animals was examined after topical application of NTZ-TEG and formalin (0.8%). The topical irritation, i.e. edema or erythema was scored and calculated according to the Draize scoring method [A skin irritation experiment was carried out according to the procedure given by Draize et al. The animg method . The prig method . The stu. 2.3.10In vitro anti-leishmanial activity was carried out by using diphenyl-tetrazolium (MTT) colorimetric assay. The parasites were incubated in the M-199 medium having 1% FBS, 100 IU ml−1 penicillin and 100 µg ml−1 streptomycin-sulfates at 24°C. The promastigotes were counted in a Neubauer hemocytometer and were seeded at the concentration of 1 × 106 promastigotes ml−1 in each well of a 96-well plate having 20 µl samples, at the concentration of 100 µg ml−1 (having less than 1% dimethyl sulfoxide (DMSO) in PBS). DMSO (1%) and amphotericin B (10 µg ml−1) in PBS were employed as negative and positive controls, respectively. Incubation of these culture plates was done for the period of 72 h at a temperature of 24°C. After this the sterilized, pre-filtered MTT solution at a concentration of 4 mg ml−1 in distilled water was prepared, added to the culture plates and incubated for 4 h at 24°C. After that, the supernatant was carefully removed without disturbing the sediment having formazan crystals. For the dissolution of formazan crystals, DMSO (100 µl) was added in the sediment. After 1 h, the absorbance was measured at the wavelength of 540 nm using the microplate reader. The half-maximal inhibitory concentration (IC50) was calculated by using GraphPad prism software [software . The persoftware :growth i. 2.3.114 million cells per well and incubated for 24 h with 5% CO2 supply at 37°C [The resident macrophages were collected from the peritoneal cavity of the rats by injecting the cold RPMI (cRPMI) media. Moreover, the media containing these macrophages was withdrawn slowly and placed on ice. The collected suspension was centrifuged at 4°C for 10 min. The obtained pellets were then dispersed in the cRPMI media containing 10% FBS and 3% penicillin/streptomycin. The cells were counted using a hemocytometer and placed in the 96-well plate at the concentration of 2 × 10 at 37°C .−1 were added into a 96-well plate and incubated for 24 h in a controlled environment of 5% CO2 and at 37°C. After 24 h, the supernatant was removed, and macrophages were washed twice with sterile PBS to remove the free drug. The attached macrophages were scraped off and centrifugation of cellular suspension was carried out at 4°C for 5 min to obtain pellets of macrophages. Methanol was added and sonicated for 5 min. The UV- spectroscopic examination was done to determine the NTZ content up taken by the macrophages [For the cell uptake experiment, the harvested macrophages were employed. The macrophages were washed with sterile PBS to remove the non-adherent cells. After that, NTZ-dispersion and NTZ-TES at the concentration of 50 µg mlrophages .. 2.3.12The stability study was conducted at temperatures 4 ± 2°C and 30 ± 2°C for a period of four months to evaluate the effects of different storage conditions on the optimized formulation. Samples stored at respective temperatures were evaluated for PS, PDI, ZP and the physical stability .. 3. 3.1esign Expert® . The Box-Behnken design was applied specifically because of lesser runs generated than a central composite design, analysing three to four variables. It is preferred if the goal is to accurately optimize a process using a minimum number of experiments in less time and thus provide a far more efficient and cost-effective technique than the conventional processes of formulating and optimization of dosage forms [For the optimization of blank TES, a two-level, three-factors Box-Behnken design was used by employing the software Dge forms . In tablp < 0.05. ‘Adequate precision’ was calculated for each response in order to confirm that the given model can navigate the design space and it was observed that the signal to noise ratio was adequate as all the values were greater than 4. Moreover, ‘predicted R2’ values and ‘adjusted R2’ values were reasonable in agreement for all the responses, i.e. difference is less than 0.2 [The regression analysis for all the responses i.e. PS, PDI and ZP was done by the software shown in the electronic supplementary material, table S2. The obtained results from regression analysis indicated that the models produced for each response were significant with than 0.2 . The mag. 3.2esign Expert® was employed for the data analysis of every response and ANOVA was employed to estimate the effect of each factor on all responses along with the p-values. p < 0.05 indicates results that are significant. The effect of independent variables i.e. PL90, S80 and ethanol on PS of the TES is shown in figure 2a. The three-dimensional plots showed that by changing the ethanol concentration there was a significant (p < 0.0001) effect on the PS, and it produced a negative effect on PS, i.e. by increasing the ethanol concentration there was a significant reduction in PS of TES. The reason behind this reduction was that the increased ethanol concentration causes the thinning of the vesicle membrane owing to interpenetration of hydrocarbon chains of ethanol within the lipid bilayers [p = 0.9083) reduction in PS was observed which might be owing to the softening effect of surfactant on membrane and owing to the increased elasticity of vesicular membrane [p = 0.4633) PS reduction was observed by increasing the lipid concentration, which has also been reported by previous studies. This reduction was owing to the fact that at higher concentration of lipid, more phospholipids are available which might act as a surfactant thus increasing the stabilization of vesicles and favour the formation of smaller particles [The size of a particle is crucial in terms of TES permeation from tiny skin pores. The smaller size i.e. less than 200 nm is desirable for the effortless permeation of TES from skin. The size of all prepared TES was within the desirable range i.e. ranging from 126.16 to 188.8 nm. Dbilayers . Moreovebilayers . On the membrane ,64. Moremembrane . Similararticles ,67.Figu. 3.3PDI indicates the homogeneity of the formulation. The higher PDI indicates the heterogeneous PS of the nanoparticles which in turn may affect the stability of the formulation. The PDI of the blank TES ranges from 0.075 to 0.185 which is within the optimal range.b indicated that there was a negative correlation between the ethanol and PDI. By increasing the ethanol concentration, a significant (p < 0.001) reduction in PDI was observed. Similar results were reported previously by Nayak et al. [p = 0.0952) increase in PDI was observed, the same effect was also reported in a previous study [p = 0.026) increase in PDI was observed by the increasing lipid concentration which was probably owing to the increase in number of the non-uniform size vesicles formed as a result of increased lipid concentration [The three-dimensional graphs shown in figure 2k et al. . This isk et al. . The effus study . A signintration .. 3.4For the long-term stability of nanoparticles, ZP values must be in optimal range. The particles with ZP more than ±60 mV have excellent stability properties while less than ±10 mV indicates poor stability properties resulting in rapid agglomeration of the nanoparticles. The ZP of the blank TES ranges from −45 to −15.4 mV which is in the optimal range.c indicated that by increasing the ethanol concentration the ZP of TES significantly decreased with p < 0.0001. The high content of ethanol produces a negative charge on the polar heads of the phospholipids resulting in electrostatic repulsion. This repulsion not only prevents the aggregation of nanoparticles and promotes the stability properties [p = 0.2601) increase in the ZP was observed by increasing the surfactant concentration [p = 0.0762) [The three-dimensional plots shown in figure 2operties  but alsooperties . On the ntration . The reantration . The inc 0.0762) .. 3.5After loading the different concentrations of NTZ in optimized blank TES, the physical unstability in the form of precipitation and reduced %EE was observed which might be owing to the partial lipid bilayer solubilization in high ethanol content, resulting in leaky vesicles leading to the loss of drug and thus reduced %EE . MoreoveTEM analysis of NTZ-TES showed that the particles were spherical in shape, sealed and uniformly distributed with a smooth outer bilayer indicating lower PDI . Moreovea) indicated a characteristic peak of N-H stretching at 3356.14 cm−1 and aromatic C-H stretching at 3090.12 cm−1. The other characteristic bands at 1770.65 cm−1 and 1523.76 cm−1 are indicative of the C = O and N = O stretching, respectively [figure 4b, the absorbance band at 2924.09 cm−1 indicated the O-H stretching, while the characteristic peak at 1172.72–1247.94, 968.27 cm−1 and 1090.71 showed the P=O, N(CH3)3 and P-O-C stretching, respectively [−1 indicates the C=O stretching of the phospholipid [figure 4c,d), all the characteristic peaks of NTZ and PL90G were intact indicating no interaction between drug and excipients. The only change in intensity of the characteristic peak of PL90G at 1734.01 cm−1 (C=O) in NTZ-TES spectra was owing to the formation of H-bonds between the OH-groups and carbonyl groups of the phospholipid. Furthermore, this intensity change in peak is the indicator of change in the relative number of the free and hydrogen-bonded C=O groups. The band at 1463.97 cm−1 is an indicator of the -CH2- group of the fatty acid chains of the lipid and the span [−1 and 1247.94 cm−1 to 1089.78 cm−1 and 1265.3 cm−1 in NTZ-TES spectrum was the indicator of the interference caused at the polar heads of the PL90G in the TES [The FTIR spectrum of NTZ a indicatectively . On the . 3.6On physical inspection, the NTZ-TEG was clear opaque, homogeneous in texture, and pale yellow in colour owing to NTZ. The pH of the NTZ-TEG was 5.02 ± 0.11 , which i. 3.7± 26.46% and 313.33 ± 18.92%, respectively, which indicated the good spreadability of the gel [In order to evenly and homogeneously apply the gel on skin, it must have good spreadability . The spr the gel .. 3.8in vitro release of NTZ from NTZ-dispersion, NTZ-TES and NTZ-TEG was analysed at pH 5.5  [a), while in the case of the NTZ-TES and NTZ-TEG, the release of NTZ was low and prolonged in comparison with the pure drug dispersion. After 3 h, approximately 30% drug was released from NTZ-TES and 15% from NTZ-TEG, while after 24 h of the study almost 40% drug release was observed in case of NTZ-TES and 22% drug was released from NTZ-TEG, indicating a slower and prolonged release profile. In the case of NTZ-TEG, the drug release rate was much lower than the NTZ-TES, because the drug has to cross two barriers, i.e. first to release from the TES and second from the chitosan gel matrix. The data of drug release was subjected to different release kinetic models in order to understand the mechanism/model which was being followed for drug release from TES and gel . The Korsmeyer-Peppas model showed highest R2 values for both the NTZ-TES and NTZ-TEG. Moreover, the values of diffusion coefficient (n)  for both NTZ-TES and NTZ-TEG were less than 0.5 indicating that the release followed the quasi-Fickian diffusion mechanism [The omal pH) . The relechanism .Figure . 3.9ex vivo permeation experiment was conducted for NTZ-dispersion, NTZ-TES and NTZ-TEG at pH 7.4 using the Franz diffusion cell. The per unit time cumulative concentration of NTZ permeated was estimated by plotting the cumulative amount (µg cm−2) on the y-axis and time (h) on the x-axis as shown in figure 5b. The penetration rate of NTZ-TES was significantly higher than NTZ-TEG and NTZ-dispersion . Within 1 h, 69.84 µg cm−2 of NTZ-TES was permeated while 34.69 µg cm−2 of NTZ was permeated from NTZ-TEG. On the other hand, only 6.9 µg cm−2 of NTZ was permeated from plain NTZ-dispersion within 1 h, which was further increased with the passage of time owing to the mild skin irritation and disruption caused by the pure NTZ-dispersion, also confirmed by the histopathological study of skin as shown in figure 5c. The permeation of drug from the NTZ-TEG was slower in comparison with the NTZ-TES. The permeability coefficient and flux of NTZ-TES were approximately seven and five times higher than the NTZ-dispersion, while for the NTZ-TEG flux and Kp were approximately three and five times higher than the plain drug dispersion, respectively. Thus, the overall results proved that NTZ-TES have high skin penetrating potential in comparison with the pure drug. This high penetration rate of the NTZ-TES might be owing to its ultra-deformable nature and increased fluidity of membrane imparted by high ethanol content. TES have both the edge activator-like TES and a high ethanol percentage just like ethosomes, thus it has properties of both the vesicles and also shares the mechanism of penetration of both transferosomes and ethosomes [An thosomes .a, the NTZ-TES and NTZ-TEG showed higher drug retention in skin layers as compared to the NTZ-dispersion, and the deposition of NTZ was higher in the dermal layer of skin in comparison with the SC layer. The observed results proved that by using TES deeper skin layer permeation can be achieved, and the deposition of TES in the dermal layer provides the prolonged supply of NTZ for enhanced therapeutic effect and reduced systemic adverse effects [After 24 h of skin permeation study, the skin was removed, and a skin deposition study was performed. The results of the skin deposition study showed a higher retention of NTZ-TES in the dermal layer of the skin which was owing to its highly deformable nature along with promoting effect of ethanol on penetration. The ethanol in TES when applied to skin increases the fluidity of lipid bilayers of SC, thus enhances the deeper skin penetration of TES . As showfigure 6 effects .Figure . 3.10−1 and 3228.73 cm−1 in the untreated and NTZ-TEG-treated skin, respectively, confirmed that the hydrocarbons of the skin lipid bilayer were intact, and no damage was caused by the NTZ-TEG after their topical application. However, in case of NTZ-dispersion-treated skin, this peak was shifted to 3428.03 cm−1 indicating disturbance in lipid bilayer of the skin figure 6b. Moreover, absorbance bands at 2970.01 cm−1 and 2811.05 cm−1 in case of untreated skin indicated the symmetric and asymmetric vibrations of CH2 bonds of the hydrocarbons of lipids. However, in case of NTZ-TEG, the intensity of peaks at 2977.22 cm−1 and 2811.08 cm−1 was reduced in comparison with the untreated skin, indicating the slight extraction of lipids from the SC by TES. On the other hand, the skin treated with NTZ-dispersion showed no peaks of symmetric and asymmetric CH2 bonds of lipids, indicating the complete extraction of lipids of the SC layer of skin. Untreated skin showed peaks at 1650.22 and 1511.10 cm−1 which indicates the amide bonds of proteins of the SC, i.e. C=O stretching and C-N stretching, respectively. In case of NTZ-TEG, the peaks of the amide bonds i.e. 1650.01 and 1511.22 cm−1 were intact indicating no change in protein structure, while in case of NTZ-dispersion, a significant reduction in intensity of peaks at 1650.13 and 1511.02 cm−1 was observed which indicated the interaction of NTZ with the proteins of the skin [The FTIR of the skin was performed in order to assess the structural changes after treatment with NTZ-TEG and NTZ-dispersion. FTIR analysis of untreated, NTZ-TEG-treated and NTZ-dispersion-treated rat skin was performed. The peaks at 3228.03 cmthe skin . The res. 3.11in vivo skin irritation study showed that the PDII of NTZ-TEG was 0.4 in comparison with a standard irritant, i.e. 0.8% formalin indicated a PDII of 4 . The obtained score of less than 2 indicates the non-irritant and good safety profile of the NTZ-TEG in comparison with the standard skin irritant. The results of histopathological study indicated that the epidermal layer was intact and no signs of infiltration or loose collagen fibres were observed in case of skin treated with NTZ-TEG in comparison with the standard skin irritant. The histopathological results of NTZ-TEG-treated skin were the same as the normal or untreated skin histology as shown in Skin irritation limits the use and patient compliance in case of topical formulations, thus the topical gel must be non-irritant and safe to apply ,87. A sk. 3.12in vitro anti-leishmanial assay against the promastigotes indicated a mean percentage inhibition of 51.99 ± 1.29% in case of NTZ-TES which was significantly higher in comparison with the NTZ-dispersion. The IC50 values of 13.4 ± 1.76 and 6.9 ± 0.29 µg ml−1 were obtained for NTZ-dispersion and NTZ-TES, respectively  NTZ was assimilated by the macrophages treated with plain drug dispersion while in the case of NTZ-TES, 12.8 ± 0.32 µg (25.6%) NTZ was recovered from the lysis of 2 × 10rophages ,89.. 3.14Stability study was performed for the NTZ-TES for a period of four months at room temperature and refrigerating temperature. The nanoparticles were characterized with regard to physical stability, PS, PDI and ZP. The results indicated non-significant variation in ZP, PS and PDI at temperature of 4 ± 2°C as shown in . 4in vitro release study of NTZ-TES and NTZ-TEG showed a prolonged release behaviour and mechanism of release was diffusion controlled. The ex vivo skin permeation study of NTZ-TES and NTZ-TEG indicated a higher permeability of TES to deeper skin layers and enhanced retention in the dermal layer of skin for local effect. The in vivo skin irritation and histopathology study of NTZ-TEG showed a safe and non-irritant behaviour of NTZ-TEG. The higher percentage inhibition and lower IC50 value for NTZ-TES against promastigotes of L. tropica indicated its efficacy against CL. The macrophage targeting performance of NTZ-TES was confirmed by the quantitative cellular uptake study. In the light of overall results, it is concluded that the NTZ-TEG can be effectively used for the topical delivery of NTZ in CL.The NTZ-TES were successfully prepared and optimized in terms of PS, PDI, ZP, TEM and FTIR. The optimized NTZ-TES were nano-sized, having low PDI, spherical in shape, and no interaction was found between the excipients of TES. The characterization of NTZ-TEG indicated uniform consistency, optimal pH for skin application, good spreadability, desirable viscosity and shear thinning nature of the gel which were the desired characteristics for topical gel application. The


Studies have shown an increasing prevalence of mental health issues in the general population during the COVID-19 pandemic. Among them, pregnant women are a specific population at particular mental risk.The objectives of our study were to assess dysfunctional anxiety in women recovered from COVID-19 and to identify the impact of pregnancy on coronavirus-related dysfunctional anxiety.This was a cross-sectional case-control study.The women in the case group have been infected with Sars-Cov 2, with a benign or pauci-symptomatic clinical form, and cured for one to two months at the time of the study without any post-COVID complications. Women included in the control group have not been infected with Sars-Cov 2 .Anxiety was assessed by the Coronavirus Anxiety Scale (CAS).In total, we recruited 30 women in the case group and 30 women in the control group.The average age of the case group was 35.8 ±6.8 years versus an average age of 35.3 ± 6.33 years in the control group. In each group, four women were pregnant (13.3%). Nearly one-third of the patients in the case group had a CAS score indicating dysfunctional anxiety probably related to coronavirus (33.3%), with a significant difference with the control group (p=0.026). In the case group, pregnancy was a risk factor for dysfunctional anxiety with p=0.036, OR=19.46 and CI95% = [1.21-314.00].COVID-19 has a negative impact on perinatal mental health. Specific support for pregnant women is recommanded during the COVID-19 pandemic.No significant relationships.


Obesity is an important public health problem nowadays. Long-term obesity can trigger a series of chronic diseases and impair the learning and memory function of the brain. Current studies show that scientific exercise can effectively improve learning and memory capacity, which also can provide benefits for obese people. However, the underlying mechanisms for the improvement of cognitive capacity under the status of obesity still need to be further explored. In the present study, the obesity-induced cognition-declined model was established using 4-week-old mice continuously fed with a high-fat diet (HFD) for 12 weeks, and then the model mice were subjected to an 8-week swimming intervention and corresponding evaluation of relevant indicators, including cognitive capacity, inflammation, insulin signal pathway, brain-derived neurotrophic factor (BNDF), and apoptosis, for exploring potential regulatory mechanisms. Compared with the mice fed with regular diets, the obese mice revealed the impairment of cognitive capacity; in contrast, swimming intervention ameliorated the decline in cognitive capacity of obese mice by reducing inflammatory factors, inhibiting the JNK/IRS-1/PI3K/Akt signal pathway, and activating the PGC-1α/BDNF signal pathway, thereby suppressing the apoptosis of neurons. Therefore, swimming may be an important interventional strategy to compensate for obesity-induced cognitive impairment. Obesity, as a sub-health stage with the excessive accumulation of body fat, is mainly affected by genetics and lifestyles. Due to the long-term chronic inflammatory state and abnormal metabolism as the hotbed for metabolic, endocrine, cardiovascular, and nervous system diseases, obesity has become a public health problem that is a current research focus all over the world. Currently, nearly one-third of the world’s population is overweight or obese . AccordiThere are many ways to intervene obesity, including exercise, diets, drugs, and surgery. Previous studies have demonstrated that obesity-induced impairment of cognitive and memory function can be suppressed and rescued after exercise , ketogenIt is well known that scientific and reasonable exercise can significantly promote the health of the body and reduce the occurrence of sports injuries. Relevant studies have shown that swimming can effectively improve learning and memory functions and increase hippocampal BDNF level . TherefoThirty 4-week-old specific pathogen-free (SPF) grade C57BL/6 male mice (body weight: 19.9 ± 1.4 g) were purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention (Certificate No. 42010200005338). The mice were randomly divided into the regular diet group (10 mice) as the normal control (NC) group and the HFD group (20 mice) to establish the obesity model. The obese mice were then divided into two subgroups, which were the obesity-control (OC) group without swimming and the obesity-exercise (OE) group with loading-free swimming for 8 weeks A. The miThe obese mice were subjected to non-intermittent swimming training in a swimming tank (30 ± 1 °C) without weight bearing for 8 weeks, five times a week and 60 min each time. In order to relieve the stress from swimming, the mice were provided with incremental exercise time for adaptation from 15, 20, 30, 45, and 60 min in the first week. The swimming time was set up in the early morning in accordance with the mouse biological clock (18:00–20:00). During the exercise intervention in this study, all mice were abstained from other additional physical stimulations.After the swimming intervention for 8 weeks, all mice were subjected to a new object recognition test for evaluating the learning and memory function. The mice to be tested were placed in the test environment for 24 h in advance for environmental adaptation, and the training began to place two identical objects on the same side. The mouse was placed in the field with its back facing the two same objects, and the heads of the mice were at the same distance from the two objects. After the mice were put into the test environment for 10 min to adapt to the test environment, the mice were immediately put back into the rearing box, and after 1 h of rest, one of the objects was replaced with another object with a different color and shape to conduct new object recognition tests within 5 min, as shown in the general process B,C.After the novel object recognition test, 3 mice in each group were sequentially perfused with 0.9% saline and 4% paraformaldehyde (pH 7.4) under anesthesia. After perfusion, the mice were sacrificed by the dislocation of cervical vertebrae, and the brain was removed and placed in 4% paraformaldehyde at 4 °C overnight for paraffin embedding and staining. The paraffin block of mouse brain tissue in each group was cut coronally into sections with the thickness of 5 μm using a microtome, and the brain tissue sections were then subjected to de-paraffinization and rehydration, Nissl staining, dehydration sealing, and microscopic examination. HE staining and antigen retrieval were performed after de-paraffinization and rehydration, endogenous peroxidase blocking, primary antibody incubation at 4 °C overnight, secondary antibody incubation at room temperature, color development, hematoxylin staining, dehydration, clearing, mounting, and microscopic evaluation. In immunofluorescence, the primary antibody and corresponding secondary antibody, CY3-TSA, were added for the probing. Nuclei were counterstained with DAPI. The images were acquired by an imaging system  and fluorescence was analyzed using ImageJ software .g for 5 min at 4 °C. After protein concentration of the supernatant was detected by the BCA method, the aliquots of the supernatant were mixed with 2× loading buffer and then subjected to a metal-plate bath at 95 °C for 5 min to denature proteins. Approximately 25 μg of total protein in the prepared samples were separated using 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The target protein in the membrane was probed by a specific primary antibody against IL-6, TNF-α and BDNF , p-IRS-1ser307, IRS-1, p-Aktser473, Akt, p-PI3K, PI3K, JNK, p-JNK, PSD95, and β-actin , as well as the corresponding secondary antibody . Protein bands were visualized by using an enhanced chemiluminescence (ECL) reagent and imaged using an ultra-sensitive fluorescence/chemiluminescence imaging system ChemiScope6300 .The hippocampal tissues of the mice were collected on a low-temperature plate and placed in liquid nitrogen immediately and then transferred to a −80 °C refrigerator for storage and future use. Hippocampal tissue samples were subjected to pre-cooled cell lysis in the presence of protease inhibitors and to homogenization in ice for 30 min. The supernatant was harvested by centrifuging at 10,000× p < 0.05.All data were expressed as mean ± standard deviation (M ± SD). Statistical analysis was conducted by GraphPad Prism software . A one-way analysis of variance (ANOVA) was used to analyze statistically significant differences between multiple groups for parametric data. Otherwise, a nonparametric Kruskal-Wallis analysis was performed. The significant difference was considered at p < 0.001) from 6 to 12 weeks and extremely significant differences in body weight from 4 to 20 weeks (p < 0.001), indicating that HFD can rapidly promote the body development of the mice. After obesity modeling for 12 weeks, the obese mice were divided into the obesity-exercise (OE) group and the obesity-control (OC) group. Compared with the OC group, the body weights of the mice from the OE group showed a significant decrease after a swimming intervention for 8 weeks (p < 0.05)  B. Therefp < 0.05). In contrast, after a swimming intervention for 8 weeks, the learning and memory capacity of the mice from the OE group was significantly higher than that in the OC group (p < 0.05)  C. These Long-term consumption of a high-fat diet may lead to the damage and functional decline of hippocampal neurons . In ordep < 0.05); on the contrary, swimming intervention reversed the obesity-induced increase of IL-6 and TNF-α   B,C. The  < 0.01) D, indicaser307/IRS-1 ratios in hippocampal tissues of the mice from the OC group revealed a significant increase (p < 0.05); in contrast, p-PI3K/PI3K and p-Aktser473/Akt ratios exhibited an obvious decrease (p < 0.05), suggesting that a high-fat diet contributes to inflammation-induced insulin resistance. On the other hand, the swimming intervention suppressed the increase of p-JNK/JNK and p-IRS-1ser307/IRS-1 ratios (p < 0.05) and the reduction of p-PI3K/PI3K and p-Aktser473/Akt ratios   B–D, therp < 0.05, p < 0.01), while a swimming intervention could rescue the down-regulation of these proteins . Therefore, a swimming intervention is beneficial to the rescuing of down-regulated PGC-1α, BDNF, and PSD95 in hippocampal tissues of the mice caused by the long-term consumption of a high-fat diet, thereby enhancing neurotrophic factors and synaptic plasticity.The reduction of neurotrophic factors and synaptic plasticity in hippocampal tissues of the mice with obesity may be the inducers for poor learning and memory capacity . To undep < 0.05); on the contrary, a swimming intervention could up-regulate Bcl-2 and down-regulate Bax to execute the suppression of hippocampal neuronal apoptosis (p < 0.05). These results suggest that the long-term consumption of a high-fat diet may trigger apoptosis of the hippocampal neurons, and swimming can rescue this phenomenon to some extent.A high-fat diet can induce the oxidative stress for stimulating apoptosis of the hippocampal neurons in mice, thereby triggering a reduced learning and memory capacity . To furtpoptosis A–C. Althpoptosis D (p < 0.Obesity can lead to the impairment of learning and memory capacity, which is confirmed by increasing studies. However, the molecular mechanisms for the impairment of learning and memory capacity caused by obesity are unclear. The 1–6-months-old C5BL/6 mice are equivalent to 12–30-year-old humans during the rapid development period to adult period . TherefoThe occurrence of obesity may lead to a significant decline in memory, but some studies have found that the decline in cognitive function caused by a high-fat diet may precede the occurrence of obesity . RelevanFrom the point of view of molecular mechanisms, the impaired learning and memory capacity may be related to the increased level of hippocampal inflammation caused by high-fat diets . It has Inflammation caused by obesity may be an important factor associated with insulin resistance . PreviouIn addition, neurotrophic factors and synaptic plasticity are closely related to improving learning and memory functions. In the present study, the expression levels of PGC-1α, BDNF, and PSD95 showed a downward trend in the hippocampal tissues of obese mice, as consistency with the literature reports describing the alleviation of learning and memory impairment caused by soybean oil-induced BDNF reduction . As an iThe long-term consumption of high-fat diets may be detrimental to the survival of hippocampal neurons, and corresponding studies have shown that exercise interventions can promote the improvement of hippocampal function by suppressing the apoptosis of hippocampal neurons, thereby enhancing learning and memory capacity . ExercisAs is well known, dietary modification is another important way to mitigate obesity and enhance learning and cognitive capacity. In order to better understand the effect of exercise intervention on regulating the progression of obesity, in our study, the benefits of exercise intervention were significantly highlighted. Swimming intervention can alleviate inflammatory responses, promote insulin sensitivity, up-regulate neurotrophic factors, increase synaptic plasticity, and suppress apoptosis A, as welAdolescents require more nutrients and energy, but the consumption of long-term high-fat diets can also lead to body weight gain and impaired learning and memory capacity, which is highly correlated with neuroinflammation, insulin resistance, reduced neurotrophic factors, and increased neuronal apoptosis. Swimming intervention can reverse these abnormal changes to rescue the impaired learning and memory capacity in obese mice by reducing obesity, alleviating hippocampal neuroinflammation, activating insulin signaling, promoting the generation and secretion of neurotrophic factors, and suppressing hippocampal neuronal apoptosis in adolescent mice, with the involvement of the JNK/IRS-1/PI3K/Akt and PGC-1α/BDNF signal pathways .


In the present study, p-hydroxybenzoic acid-grafted chitosan (PA-g-CS) conjugates with different grafting degrees were synthesized by a free radical-regulated grafting approach. The conjugates were further developed into films by casting, and their characteristics and preservative effects on fresh-cut jackfruit were evaluated. Compared to the CS film, the PA-g-CS film showed comprehensive performance improvements, including enhancements of water solubility, anti-ultraviolet capacity, antioxidation, and antibacterial activity. Moreover, compared with CS film, some appreciable and favorable changes of physical properties were observed in the PA-g-CS films, which included water vapor permeability, oxygen permeability, surface morphology, moisture content, and mechanical intensity. Furthermore, compared to CS alone, the application of PA-g-CS films to fresh-cut jackfruit exerted a beneficial effect on the quality of products, as indicated by the inhibition of weight loss, softening, and membrane damage, the maintenance of soluble solids and ascorbic acids contents, as well as a reduced bacterial count and a higher sensory score. Among these PA-g-CS films, the best preservation effect was achieved with the highest degree of grafting (PA-g-CS III). The results suggested that the PA-g-CS film has the potential to be explored as a new type of packaging material for the preservation of fresh-cut fruits and vegetables.  Artocarpus heterophyllus L.) is one of the world’s largest fruits, and each mature tree may produce about 10 to 200 individual fruits [Jackfruit  is obtained by the deacetylation of chitin, and it is considered an excellent biomaterial for edible coatings. Manufacturing food packaging film is beneficial and practical due to its nontoxicity, film-forming ability, biocompatibility, biodegradability, antimicrobial activity, and antioxidative activity ,9,10. HoP-hydroxybenzoic acid (PA), a monohydroxy phenolic derivative of benzoic acid, is widely used as an antioxidant, preservative, and fungicide in foods, beverages, medicines, and cosmetics ,24. Our 2O2) redox system in an inert atmosphere. Then, the PA-g-CSs with varying grafting ratios were cast into the films. Later on, the physical properties , mechanical behaviors (elongation at break and tensile strength), barrier performance , biological activities , and surface morphologies of the prepared PA-g-CS films were evaluated for the first time. Finally, we applied the films onto the fresh-cut jackfruit and evaluated the preservative effects of chitosan and PA-g-CS films.However, to our knowledge, no study has investigated the impact of PA-g-CS based film on the preservation of fresh-cut jackfruit during storage. In this study, a free radical-mediated method was used to graft PA onto CS, which was accomplished using an ascorbic acid (Vc)-hydrogen peroxide (H2O2 (99% purity) were provided by Aladdin Bio-Chem Technology Co., Ltd. . All remaining reagents were analytically pure. Jackfruits at 80% maturity (light bluish green) were picked from a commercial farm in Haikou, China, and the fruits with uniform sizes were selected for the study. Chitosan with a 95% deacetylation degree and a dynamic viscosity coefficient of 100 to 200 mPa was provided by Macklin Biochemical Co., Ltd. . Folin–Ciocalteu reagent (98% purity), p-hydroxybenzoic acid (95% purity), ascorbic acid (99% purity), and 30% Hv/v). Later, varying amounts of PA were added to the above mixture, resulting in different CS-to-PA molar ratios of 1:0.3, 1:0.7, and 1:1. Simultaneously, 0.2 g of ascorbic acid (Vc) was added to all the reactors. The reactor was then injected with oxygen-free nitrogen gas for 30 min under stirring conditions, followed by the addition of 1 mL of 30% H2O2 solution. This was incubated for 12 h in an oxygen-free nitrogen atmosphere. Later, distilled water was used to dialyze the above reaction mixture for 72 h using a membrane  to eliminate the unreacted phenolic acids and additional soluble substances. Finally, PA-g-CS was obtained by lyophilizing the dialysate with different ratios, which were referred to as PA-g-CS-I, PA-g-CS-II, and PA-g-CS-III, respectively. The cold trap temperature of the freeze dryer  was −30~−40 °C, the sublimation temperature was 50~60 °C, and the time was 8–10 h. The content of polyphenols in the product was detected using the Folin–Ciocalteu method. The degree of grafting was determined to be 35.16 ± 2.07, 65.02 ± 2.34, and 101.16 ± 4.46 (mg PA/g PA-g-CS), respectively.The preparation of PA-g-CS was carried out according to our previous method . Here, 1w/v) was dissolved in 150 mL of acetic acid aqueous solution (1% v/v). Then, this solution was mixed with 50% (on a dry-weight basis of PA-g-CS) glycerol plasticizer and stirred for 60 min at 25 °C. The resulting mixture was ultrasonicated for 30 min at 200 W and 40 kHz for air bubble elimination. Later, the solution was poured into a 24 cm × 24 cm size glass plate and allowed to dry for 48 h at 40 °C. Finally, the dried PA-g-CS films were removed from the plate and stored at the relative humidity (RH) of 50 ± 2% and a temperature of 25 °C for a minimum of 48 h. Similarly, the control CS films were also prepared.First, PA-g-CS  in the range of 4000–400 cmOnce the film surfaces were fractured with liquid nitrogen soaking, they were coated with a thin layer of gold. A S-4800 SEM system  was utilized to examine the surface morphologies of the film samples at an accelerating voltage of 10 kV.A represented absorbance of the film at 600 nm, while X represented the thickness of the film (mm). Accordingly, the opacity results were presented as A600/mm.Each rectangular film sample (1 cm × 4 cm) was attached tightly to the inner wall of the cuvette for the measurement of optical transmittance with a double beam spectrophotometer  at 200–800 nm. An empty cuvette was used as the control. Film opacity was calculated based on the 600 nm absorbance by the following formula :Opacity W0 (g) was the initial equilibrium weight of the film (2 cm × 2 cm), while W1 (g) was the weight after the film was dried at 105 °C for 24 h.In this study, MC was referred to as the weight loss percent of the film after 24 h of drying at 105 °C. The computational formula used to calculate the MC value was as follows :MC(%)=W0W0 (g) was the initial equilibrium weight of the film (2 cm × 2 cm), while W1 (g) was the undissolved substance weight following 24 h of film immersion.The percentage of the dissolved substance following 24 h of thorough film immersion in distilled water was referred to as WS. The computational formula used to calculate the WS value was as follows:C represented the slope of the temporal graph of weight gain (g/s), x denoted the thickness of the film (m), A denoted the permeation area (m2), while ΔP was the difference between the partial vapor pressure of pure water and dry atmosphere with a value of 2339 Pa at 20 °C. The WVP results were presented as g/(m·s·Pa).To measure the WVP of films, Liu et al.’s  method wOP for the film was as follows:m1 represented the final weight of the bottle following 48 h of equilibration, m0 represented the initial bottle weight, t denoted the equilibration duration(s), and A denoted the exposed surface area of the film with a value of 0.00049 m2.Determination of the OP was accomplished as per the procedure found in the literature . Every 3EAB) and tensile strength (TS) of the films [A tensile tester  was used to measure the elongation at break  and UV-irradiated for 30 min in a laminar flow cabinet. The puncher was used to prepare an 8 mm diameter film of circular shapes, which were then subjected to 30 min of UV irradiation for disinfection. These films were then transferred onto the sterilized Petri dishes. Subsequently, the film samples were placed onto the inoculated agar plates in the laminar flow cabinet using sterilized forceps. The samples were then allowed to stand at room temperature for 30 min to diffuse the bioactive compounds from the films to the medium. The plates were incubated for 24 h at 37 °C. Finally, the clear zone diameter at the periphery of the film was measured and recorded in mm, including the diameter of the disk.To determine the antibacterial activity of the film samples, the agar diffusion technique was employed . Here, 1The jackfruit bud was taken out using a sharp stainless steel knife and immersed in a solution of sodium hypochlorite (100 mg/L) for 30 s. The buds with a uniform shape and size were selected for packaging after being dried for 2 h at room temperature. These jackfruit buds were randomized into five groups: (1) control without packaging treatment; (2) PA-g-CS-I film packaging; (3) PA-g-CS-II film packaging; (4) PA-g-CS-III film packaging; (5) CS film packaging. The jackfruit flesh was tightly wrapped with the respective films and then placed in a 17.5 cm × 12 cm × 7 cm plastic basket. All the groups were then stored at a temperature of 15 °C with a relative humidity (RH) of 80–90%. To determine the net weight, an analytical balance was utilized to weigh the entire individual package of jackfruit bulbs on the day of the observation. The values were presented as the percentage of weight loss. The measurement of hardness of the flesh was accomplished as per Zheng et al.’s approach, with slight modifications . The maxThe jackfruit pulp was homogenized in distilled water at a ratio of 1:4. The homogenized liquid was centrifuged at a speed of 5000 rpm/min for 15 min. The TSS of the supernatant was measured with a handheld Brix meter.A total of 10 g of the sample was extracted with 3% metaphosphoric acid and the volume was supplemented up to 100 mL. Subsequently, 10 µL of the filtrate was titrated with 2.6-dichlorophenolindophenol dye (0.025%) until the distinct rose pink color persisted for 15–20 s. The results were expressed as the average value of three replicate samples. The colony counting technique was employed to examine the TBC of the jackfruit, as per the GB 4789.2–2016 of the Chinese standard. The result was expressed as log CFU/g.1) was determined after the pulps were placed in a beaker containing 20 mL of distilled water for 30 min. Finally, the conductivity (C2) was measured after 15 min of boiling and subsequent cooling at room temperature. The computational formula to calculate the electrolyte leakage rate was as follows:The membrane permeability was presented in terms of relative conductivity and measured as described by Chen et al., with slight modifications . First, The procedure for sensory evaluation was taken from Hu et al. . Panelisp-values less than 0.05 were regarded as statistically significant.All the experiments were performed in triplicate. The values were expressed as means ± standard deviation. Data were analyzed using SPSS 25. The significance of the inter-sample comparisons was determined using Duncan’s multiple range test and the analysis of variance (ANOVA); −1 (OH- and NH- stretching in the benzene ring), 2930–2870 cm−1 (C-H stretching), 1655 cm−1 , 1590 cm−1 , 1323 cm−1 , 1030 cm−1 (C-O-C), and 896 cm−1 (pyranose ring). This finding was consistent with previous reports in the literature [−1, which was related to C=O group stretching of the saturated ester. This observation indicated the occurrence of graft co-polymerization of PA (-COOH) and CS (-OH), along with the formation of ester linkages. Moreover, it was observed that the bands of the three conjugates in amide-III shifted from 1323 to 1307, with higher peak intensity than that of the CS film. This also confirmed that the grafting reaction occurred in the amino group (-NH2). terature . The thrThe surface morphology of the CS and PA-g-CS films is presented in In recent years, scholars have widely emphasized the importance of UV-protective films, since the free radicals produced from light can compromise the food quality ,37. As sp < 0.05), which decreased with the increase in grafting ratios. Meanwhile, the WS of the PA-g-CS films were prominently higher than for the CS film (p < 0.05), which increased with the increase in grafting ratio (p < 0.05). The possible reason behind the difference in the values of MC and WS between the CS and PA-g-CS films may be attributed to the drastic impairment of the hydrogen bonding due to the incorporation of PA. This leads to a change in the hydrophilicity of PA-g-CS. Similar findings have also been reported for the chitosan films grafted with protocatechuic acid [The moisture content (MC) and water solubility (WS) are the parameters that assess water sensitivity, which is one of the prominent issues with the films used for food packaging. According to uic acid , salicyluic acid , and cafuic acid .p < 0.05), whereas the WVP values in the PA-g-CS-II and PA-g-CS-III films showed a significant decrease. The WVP value of the PA-g-CS film showed a negative correlation with the grafting rate (p <0.05). There were several reasons behind the differences observed in the WVP values. First, as shown by SEM, different films had different gaps, leading to different WVP values, and second, the hydrophilic groups were reduced by the covalent bonding between CS and PA, causing the reduction in WVP. Besides, the inner architecture of the CS film might also be hindered by the huge benzene rings in the PA moieties [The WVP of the film represents the barrier property of the packaging material towards moisture. According to moieties . In a stmoieties , a negatmoieties , the WVPp < 0.05). With an increase in the ratio of PA grafting, the OP value of PA-g-CS tends to decrease (p < 0.05). The difference in the OP value was related to the surface morphology of the films.Oxidation is a process occurring in the presence of oxygen, which can induce alteration in food properties, including color, odor, flavor, and nutritional degradation. Accordingly, the quality and shelf-life of foods can be improved by adequate oxygen blocking measures . As showp < 0.05). In the PA-g-CS films, both TS and EB were associated negatively with the ratio of PA grafting, suggesting compromised mechanical performance after the conjugation of CS with PA. The probable reason for this may be the quantitative decrease in the functional groups of CS by PA grafting, which makes the hydrogen bonding interaction between glycerol and PA-g-CS difficult [The mechanical behavior of a film can be represented by two major parameters, TS and EB. Here, TS refers to the endurance limit of a film that can withstand an applied tensile force, while EB reflects the stretching capability of a film . TS and ifficult . Additioifficult , Aljawisifficult , and Rivifficult  believedifficult . Howeverifficult  found thifficult . The reaThe DPPH assay was employed to examine the antioxidant activity of the various studied films. The reduction in the absorbance at 517 nm was regarded as the determinant of the DPPH radical scavenging capacities of a film. As observed in E. coli (G−) and S. aureus (G+), were used to assess the antibacterial performance of various studied films, whose results are listed in p < 0.05) antibacterial activity was noted in the PA-g-CS films, suggesting an enhanced antimicrobial performance of the CS film by the PA grafting. According to the extant studies, the antibacterial action can be strengthened by the synergistic interaction of PA and CS [S. aureus compared to E. coli. The antibacterial activity against S. aureus of the CS and PA-g-CS films was higher than that against E. coli. Which may be related to the fact that the lipopolysaccharide layer on the cell membrane of Gram-negative bacteria can effectively prevent the antimicrobial substances from crossing the cell membrane, giving it certain drug resistance [Two common pathogens, A and CS ,34. The sistance .p < 0.05). Comparatively, both CS and PA-g-CS film groups exhibited lower weight reductions in the fruit. This might be attributed to a protective barrier established by the films, which could help to limit the gas exchange, while decreasing moisture evaporation and reducing the loss of organic matter. Similar delay in weight loss behavior has been demonstrated in CS-coated pummelos [Factors such as water transpiration, respiration consumption, and the leakage of juice often lead to the reduction in the weight of fresh-cut fruits during storage. As shown in pummelos , litchispummelos , and grapummelos . p < 0.05). The softening observed for the PA-g-CS-III film group was significantly slower than the other groups (p < 0.05). Another serious concern in the preservation of fresh-cut fruit is rapid tissue softening, which decreases the textural quality and shortens the storage life of the products . As obseThe TSS content is also an important indicator that reflects the fruit flavor quality. As observed in p < 0.05) the loss of ascorbic acid content as compared to the control during storage. The PA-g-CS film treatment also significantly reduced (p < 0.05) the loss of ascorbic acid content as compared to the CS film group. The ascorbic acid content for the PA-g-CS-III film treatment of jackfruit was higher in comparison to other treatments at the end of storage.Ascorbic acid content is an important nutritional quality index in fruits and vegetables . The chap < 0.05). The total bacterial count in the CS film group was significantly higher than in the PA-g-CS films group. The total bacterial count in the PA-g-CS films group was negatively correlated with the degree of grafting. A high grafting degree of the PA-g-CS membrane could effectively reduce the colony growth in fresh-cut jackfruit.p < 0.05), while the relative conductivity of the CS film group was found to be prominently higher than the other three PA-g-CS groups (p < 0.05). In the PA-g-CS film groups, the grafting rate was negatively correlated with the relative conductivity, which showed that the CS grafted with PA could protect cells more effectively. Moreover, the higher the grafting rate, the better the effect.The membrane intactness and permeability are often represented as the relative conductivity, which is used as a common index. A higher relative conductivity signifies severe damage to cellular membranes. As shown in Fresh jackfruit flesh is golden yellow in color, full and bright in appearance, and crisp and tender in taste. PA-g-CS films are closely related to the oxygen permeability, water vapor transmission rate, UV blocking ability, etc. Compared to the CS film, the PA-g-CS films showed stronger antioxidant and antibacterial effects, illustrating positive associations of the conjugation of PA with CS. The films provide a relatively closed environment and a physical barrier for fresh-cut jackfruit, which protect the pulp from the external environment, and then effectively protect the fresh-cut jackfruit cells from oxidation and microbial destruction. Thus, the CS and PA-g-CS films could inhibit weight loss; maintain firmness, soluble solids, ascorbic acid content, and sensory quality; protect the cell membrane; and reduce colony count in fresh-cut jackfruit. The PA-g-CS film groups exhibited a more potent freshness-preservation effect than the CS film group, particularly the PA-g-CS-III film. The results of this work showed that the conjugated PA influenced the functional properties of the CS film and further affected its preservation of fresh-cut jackfruit. The conjugation of PA with CS enhanced water solubility, UV blocking ability, antioxidant ability, and antibacterial activities of the CS film; meanwhile, the moisture content and mechanical properties of the CS film were reduced by conjugated PA. Compared to the CS film, PA-g-CS films, particularly the PA-g-CS-III film, showed a better preservation effect on fresh-cut jackfruits in term of weight loss, hardness, soluble solids value, ascorbic acid content, total bacterial count, relative conductivity, and sensory score. The related mechanism requires future investigation. Phenolic acids (PA) showed a negative effect on the mechanical properties and a positive effect on the bioactivities of the CS film. Further research should focus on improving the mechanical property of phenolic acid-chitosan films, which will provide some idea for its commercial production. In conclusion, the conjugation of PA with CS is a more efficient way to improve the properties of CS film, and PA-g-CS conjugated films would have an extensive range of application in the food packaging industry.


Data on mental health improvement after cardiac rehabilitation (CR) are contradictory. The aim was to examine the mental and psycho-social health of patients admitted to our rehabilitation center following hospital treatment for acute coronary syndrome, before and after multidisciplinary CR.Outcome was measured at admission and discharge by the 36-Item Short Form Survey (SF-36), the Symptom Checklist-90 Revised (SCL-90R), the Coping Strategy Questionnaire (CSQ) and the 6-min-walking distance test. The patients’ health status was compared with norms of sex-, age- and comorbidity-matched data from the German general population. Score differences from norms were measured by standardized mean differences (SMDs); health changes were quantified by standardized effect sizes (ESs). Their importance for comprehensive assessment was quantified by explorative factor analysis.Of n = 70 patients followed-up , 79% had ≥ 3 comorbidities. At baseline, SF-36 Physical functioning (SMD = − 0.75), Role physical (− 0.90), Social functioning (SMD = − 0.44), and Role emotional (SMD = − 0.45) were significantly worse than the norm. After CR, almost all scores significantly improved by ES = 0.23  to 1.04 . The strongest factor (up to 41.1% explained variance) for health state and change was the mental health domain, followed by function & pain (up to 26.3%).Normative deficits in physical and psycho-social health were reported at baseline. After CR, at follow-up, all scores, except phobia, showed significant improvement. The comprehensive measurement of bio-psycho-social health should not be limited to depression and anxiety but include, especially, the somatization and social participation dimensions. Cardiac rehabilitation (CR) after a heart attack tends to focus more on patients’ physical health than on their psycho-social wellbeing. This study measured patients’ psycho-social health before and after CR, using specific self-rating instruments and the 6-min-walking-distance test and compared the results with normative data obtained from the general population. Before CR, patients’ physical and social function and role performance were below the population norms, whereas their other psycho-social health was comparable to the population norms. The psycho-social scales contributed more relevant information than the physical scales to the comprehensive description of patient health after CR. All physical and psycho-social health dimensions improved except for phobic anxiety. We recommend that CR programs also include comprehensive measurement of mental health dimensions (i.e. not confined to depression and anxiety) and of social function. Coronary artery disease (CAD) is responsible for 16% of the world’s deaths and the leading cause of mortality worldwide . BetweenThere are contradictory reports on the outcome, in terms of improved general health and reduction in mortality, of cardiovascular rehabilitation (CR) after acute coronary syndrome (ACS). A review of 22 randomized controlled trials (RCTs) found no differences in outcomes between exercise-based CR and no exercise control for all-cause or cardiovascular (CV) mortality . In contWhile several studies have been published on the somatic outcomes of CR, the literature on the effects of CR on mental and psycho-social health is sparse. Patient-reported outcomes, such as health-related quality of life (HRQL), are crucial for obtaining the patient’s perspective on the effectiveness of medical or lifestyle interventions . HoweverCardiovascular rehabilitation studies have traditionally focused on the affective dimensions of depression and anxiety. A high SCL-90R score for depression was found in 42% of CR patients . PsycholThis prospective pilot cohort study aimed firstly to quantify patients’ mental and psycho-social health and HRQL at admission to and discharge from inpatient, post-ACS CR and to compare the levels with specific population norms. The norms were matched by sex, age and number of comorbid conditions. The study’s second aim was to compare the relative importance of mental and psycho-social health and of physical health in the overall assessment and to examine how the different constructs interrelate.In addition to the traditional focus on the affective dimensions, depression and anxiety, special attention was paid in our study to phobia, interpersonal sensitivity, somatization, catastrophizing/coping, emotional role performance, and social functioning. The first hypothesis was that in these dimensions there would be substantial deficits in relation to the norm and clinically important improvements after CR. Our second hypothesis was that mental and psycho-social constructs would be at least as important as physical health dimensions for a comprehensive description of health at baseline.After treatment in hospital, ACS patients were referred to the Cardiology Department of the Rehaklinik Bad Zurzach, Switzerland for a 3-to-4-week comprehensive inpatient CR program between April 2018 and October 2021.The inclusion criterion for participation in the cardiac rehabilitation program was a confirmed diagnosis of ACS after initial acute hospital treatment, or the presence of CAD. Exclusion criteria were diagnoses other than ACS and the presence of severe comorbidities, namely severe cardiac insufficiency, valve surgery, and diseases such as terminal cancer. Also excluded from the study were patients with insufficient knowledge of the German language or psycho-intellectual abilities , which hinder participants’ understanding of the content of the rehabilitation program and assessment. The study was approved by the ethics committee of Aarau, Canton Aargau, Switzerland (EK AG 2008/026) and written informed consent was obtained from all study participants.Inpatient CR is a standardized program lasting a median of three weeks and is structured according to the guidelines of the Swiss Working Group for Cardiovascular Prevention, Rehabilitation and Sports Cardiology (SCPRS). The SCPRS recommends categorizing cardiac patients in at least three different levels . In ordeGroup 1 went on one-hour hiking and Nordic walking tours on hilly terrain five days a week throughout the rehabilitation program. Group 2 went on one-hour hikes on flat ground four days a week throughout the program. Group 3 patients had to be able to walk for at least 30 min on flat ground. In the therapy program, members of this group were to be trained five days a week. Additionally, the patients in groups 1, 2 and 3 had two to three ergometer training sessions per week. Group 4, to which the majority of patients were allocated at admission, were in generally poor physical shape, with reduced mobility. Initially, the focus was laid on gait exercises in individual therapies, and if the patients’ general condition permitted, training on a MOTOmed® , a motorThe standard exercise program consisted of 12–18 training sessions spread over 6 days per week. In accordance with international recommendations, the focus was on aerobic endurance training , 15. TheSociodemographic and disease-relevant data were gathered using a standardized questionnaire whose usefulness and validity have been established in several previous studies . All necThe Short Form 36 (SF-36) is the most-widely used questionnaire for the self-assessment of generic health and health-related quality of life , 17. It The Symptom Checklist-90 Revised (SCL-90R) is one of the best-established tools for assessing psychiatric syndromes; it also normative data from the German population (n = 2025) , 22. TheFrom the Coping Strategy Questionnaire (CSQ), the catastrophizing scale, consisting of six items, each with responses scaled by seven levels from “never” to “always”, was selected together with the two last items (control pain and decrease pain) , 24. TheAs an examiner-based measure of functional capacity, a 6MWD was applied, one of the most frequently used, best-validated and responsive functional performance tests , 28. TheFor ease of inter-scale comparison, the scoring of all the self-report instruments used was converted to the scaling system of the SF-36, i.e. 0 = worst health, maximum symptoms/disability to 100 = best health, no symptoms, full function. This means, by extension, that 0 = maximum and 100 = none on the scales of the SF-36 Bodily pain, CSQ Catastrophizing, SCL-90R Depression, SCL-90R Anxiety, etc. The walking distance of the 6MWD was quantified in meters. For the SF-36, more than 50% of the items had to be completed to determine a specific scale, which defines the instrument’s original “missing rule” , 19. ForScore data were described at admission (baseline) and discharge (follow-up) after CR by means and standard deviations. At both time points, the scores were compared to age-, sex- and comorbidities (present/absent)-matched population norms by standardized mean differences (SMDs). The SMD is the difference between the patient scores and the norm scores in numbers, divided by the pooled standard deviation of the patient and norm scores . The SMDBetween baseline and follow-up, intra-individual score differences were then standardized by the (baseline) effect size (ES) according to Kazis and the standardized response mean (SRM) according to Liang , 33. Thit-value*standard error) were given, to test whether the differences were statistically significantly different from zero (exclusion of zero by the 95%-CI) together with the corresponding p-value (type one error) [For all effect size parameters, ES, SRM and SMD, the 95% confidence intervals (95%-CI: parameter ± e error) . For allOn the basis of our experience with the comprehensive measurement of low back pain syndrome, factor analysis with varimax rotation was performed in order to quantify and compare the importance of the mental and psycho-social health dimensions and the physical dimensions . Factor Of the 17 scales examined, 14 were included for factor analysis to achieve finite data , 36. Excp = 0.050 with a 95% confidence interval of  [Sample sizes of n ≥ 70 make it possible to approximate the normal distribution of data generated by stochastic processes due to the central limit theorem and allow the use of parametric statistical parameters, as the SMD . At n = , 0.670) . A facto, 0.670) .Of the n = 81 patients included at baseline, 70 (86%) competed the CR program and the study’s assessment. One patient went home because of his wife’s death, 2 were re-hospitalized due to acute illness, and 8 withdrew their participation during the stay.The typical patient was male (79%), living with a partner (71%), well educated (47% college or higher), and was, on average 66.6 years old and slightly overweight with a mean BMI of 26.1 (only 12 = 17% were obese with BMI > 30.0). Comorbid conditions were frequently present (median = 4), most did not smoke (93%) and were moderately physically active: walking/cycling for a median ½ to 1 h/week, or practicing sports for a median 1–2 h/week. The median working capacity was 0 h/week spent at the working place (most participants were in fact retired) and 7 h/week spent on household chores. More than half the patients (54%) had received aorto-coronary bypass (ACBP) surgery and 43% percutaneous transluminal (coronary) angioplasty (PTA) during acute hospital care before CR.Physical health showed moderate score levels on the SF-36 (41.0–71.8) at baseline. Physical functioning (SMD − 0.75) and Role physical (SMD − 0.90) were significantly reduced compared to the norms whereas General health were better than the norm level (SMD 0.57). The level of pain (SMD 0.32) was slightly but not significantly lower than expected by the norm. The mean 6MWD was 370.1 m.In the domains of mental health and psycho-social abilities, SF-36 Social functioning (SMD − 0.44) and Role emotional (SMD − 0.45) were significantly impaired whereas Vitality was not. In the SCL-90R scales Depression, Anxiety, Phobic anxiety and Somatization, the mean score levels were also slightly lower than those of the norms but the differences did not reach statistical significance (SMDs − 0.12 to − 0.31). SCL-90R Interpersonal sensitivity (SMD 0.44) was significantly higher than its population norm. On the CSQ, relatively low catastrophizing was observed , whereas the ability to control and decrease pain showed relatively low levels of coping .The follow-up scores of most scales were higher than expected by the population norms by SMDs between 0.33 (SCL 90R depression) to 1.43 , Social Functioning (SMD 0.30), Role emotional (SMD − 0.01) and SCL-90R Phobic anxiety (SMD − 0.15).On almost all scales, statistical significant improvements were observed between baseline and the follow-up by ES between 0.23 on SCL-90R Interpersonal sensitivity to 1.04 on SF-36 Physical functioning; exception: SCL-90R Phobic anxiety with ES 0.12. Namely, depression and anxiety improved by ES 0.35 and 0.36, coping by ES of 0.46 to 0.53, Vitality by ES 0.76, and the 6MWD by ES 0.94. The corresponding SRMs showed somewhat higher but comparable levels to those of the ESs, except on the SCL-90R Depression scale.Mental health and function & pain were the dominating factors for the scores at baseline and follow-up as well as for the score differences explaining 26.3–41.1% of the variances followed by pain and function explaining 11.2–15.8%. All three models showed good fit to explain high proportions of the empiric variances (61.7–72.2%).At baseline, walking  formed a third factor but at the follow-up, both loaded together with SF-36 Role physical and Bodily pain. Coping was also an extracted factor covering constructs different from mental health and function and pain. For the score differences between baseline and the follow-up, SF-36 Social functioning together with Role emotional was here the third extracted factor (10.3%) before coping.p = 0.071; this was also true for SF-36 Bodily pain: 68.3 (30.3) versus 55.0 (26.4), p = 0.057. In all the other dimensions, the differences were small and not significant (p > 0.200). At the follow-up, the PTA group showed a slight trend to better function on both the SF-36 Physical functioning: 80.7 (19.9) versus 73.2 (12.7), p = 0.063 and the SF-36 Role physical: 72.4 (27.4) versus 61.3 (23.0), p = 0.073. On the 6MWD the score changes between baseline and follow-up showed more effect among the ACPB subjects: plus 140.1 (71.3) meters vs PTA 93.9 (62.9) (p = 0.009). In addition, the ACBP group experienced a slightly greater improvement in SF-36 Vitality: 19.3 (16.9) versus 11.0 (19.4), p = 0.063. In summary, state and changes on all specific mental and coping scales showed no statistically significant differences between the two acute treatment groups.The outcomes of the 38 patients with previous ACBP and the 30 patients having undergone PTA were compared (data not shown in detail). At baseline, SF-36 physical functioning was, in trend, better in the PTA group: mean = 58.0 (SD = 23.1) versus 47.8 (22.6), This prospective cohort study explored the HQRL of patients admitted to the cardiac department of our rehabilitation centre after initial hospital treatment for ACS elsewhere. The study focused on the mental and psycho-social domains before and after inpatient CR and compared the levels to specific population norms.Whereas, at baseline, physical and social function and role performance (SF-36) were significantly worse compared to the sex, age and morbidity-matched population norms, the scores on most of the scales of the mental and psycho-social domains were comparable or higher. SCL-90R Phobic anxiety was slightly but not statistically significantly worse than the population norm. The scores on the SF-36 General health, SF-36 Mental health, and SCL-90R Interpersonal sensitivity scales were significantly higher than the norms. By that, our first hypothesis that substantial normative deficits at admission to CR would be found in the mental and psycho-social dimensions was only partly confirmed. As depression and anxiety are particularly under-recognized in men, the predominantly male sample in our study might account for these findings .Nonetheless, even in the domains where the baseline scores were higher than the norms and also exceeded our expectations, there were significant improvements at discharge. Factor analysis revealed that mental and psycho-social health is a key domain in the comprehensive assessment of health before and after CR. It is more important than the domain of pain and function. Sensitivity analysis showed that the state and changes of health and HRQL were comparable between groups having received different types of pre-CR acute intervention (ACBP and PTA), and especially so in the mental health and coping dimensions. The only difference found was on the 6MWD, where the post ACBP improved more than post PTA group.A comparable study found a considerable number of symptoms on the SCL-90 scales of Depression, Anxiety, and Somatization among patients entering CR . HoweverAfter CR at our clinic, most patients’ health dimensions showed statistically significant improvements beyond the population norm levels (SMDs up to 1.43 for SF-36 Bodily pain) with standardized ESs up to 1.04  on physical health, 0.94 on the 6MWD, and up to 0.76  on the mental health scales. All scores on the SCL-90R (except Phobic anxiety), and on all three CSQ coping scales also showed improvement between baseline and follow-up. Broadening the measurement of mental health dimensions beyond the classical focus on depression and anxiety provided additional important information on CR after ACS.Two recent meta-analyses of SF-36 data, in which there was a partial overlap of the individual studies summarized, showed comparable score levels to our post-CR follow-up scores . There wOur second hypothesis that the mental and psycho-social domain is more important than physical symptoms/abilities was consistently true for all three factor analyses , as shown by explained variances up to 41.1% compared to function and pain, which explained up to 26.3%. Interestingly, the walking test formed a third factor at baseline .The main strength of our study consists in the fact that for two of the best tested and validated instruments for measuring HQRL, namely the SF-36 and SCL-90R, patient scores were for the first time compared to age- and sex- matched population norms. For the SF-36 the comparison extended to the presence or absence of comorbid conditions. Standardized scaling and quantification of differences to the norm and of changes between baseline and follow-up allowed comparison across various constructs. Explorative factor analysis quantified the contribution of the main construct domains to the comprehensive measurement of bio-psycho-social health.A limitation of our study was the small sample size of n = 70. However, larger sample sizes tend to overpower the effect estimates by narrowing the 95% confidence intervals. As reported in a large body of literature, changes below 0.30 (ES or SRM) are, on the group level, subjectively imperceptible, being smaller than the minimal clinically important differences . The basThis prospective cohort study showed that, compared to individually specific population norms, patients admitted to post-ACS CR evidenced baseline deficits in physical but not in psycho-social health domains. At the follow-up, after CR, significant improvements were found in all scores except in phobia. A broader, more comprehensive measurement of bio-psycho-social health should comprise not only depression and anxiety but in particular somatization and social participation. For clinical purposes, at least two mental health scales and at least one coping scale can be recommended for a comprehensive and specific assessment of health and HLRQ.


More importantly, because the wh-CQDs were synthesized without any additives, they exhibited low toxicity to Klebsiella sp. cells even at high concentrations. Moreover, wh-CQDs emitted bright blue fluorescence in Klebsiella sp. cells, indicating its strong penetrating ability. Correspondingly, the fluorescent cell sorting results also revealed that the proportion of cell internalization reached 41.78%. In this study, wh-CQDs derived from natural biomass were used as high-performance fluorescent probes for Fe3+ detection and Klebsiella sp. imaging. This study is expected to have great significance for the application of biomass carbon spots in the field of cellular imaging and biology.Natural biomass is used for facile synthesis of carbon quantum dots (CQDs) with high fluorescence, owing to its abundance, low cost, and eco-friendliness. In this study, a bottom-up hydrothermal method was used to prepare CQDs from water hyacinth (wh) at a constant temperature of 180 °C for 12 h. The synthesized wh-CQDs had uniform size, amorphous graphite structure, high water solubility (containing multiple hydroxyl and carboxyl groups on the surface), excitation light-dependent characteristics, and high photostability. The results showed that the aqueous solution of CQDs could detect Fe Biomass materials are a crucial renewable source of carbon on earth. Based on differences in structure, composition, and molecular weight, raw materials for synthetic biomass can be divided into three categories: micro-molecules derived from biomass , biomass components , and natural biomass  ,2. ApproEscherichiacoli and Staphylococcus aureus) . In. In37]. −1. For each measurement, the samples were scanned 64 times at a resolution of 4 cm−1. X-ray photoelectron spectroscopy (XPS) was used to characterize the content and robustness of various elements in the samples . Ultraviolet-visible (UV-Vis) absorption spectra were obtained using a Shimadzu 2550 UV-Vis spectrophotometer . The two- and three-dimensional fluorescence spectra and fluorescence intensities of the wh-CQDs were recorded using an F-7100 fluorescence spectrometer  with the excitation and emission slits of 5 nm. Fluorescence lifetime measurements were performed on an FluoroMax-4 time-resolved fluorescence spectrophotometer .The surface morphology of the wh-CQDs was determined using a Tecnai G2 F30 S-TWIN field-emission transmission electron microscopy  at an accelerating voltage of 200 kV. The particle size of the wh-CQDs was analyzed using image processing software Image J 1.52 and TEM micrographs, and the size distribution was calculated using Origin 8.0. Powder X-ray diffraction (XRD) analysis was conducted using a Bruker D8 Advance diffractometer  with Cu Kα radiation and a scanning scope of 2θ = 10–90°. The functional group information of the wh-CQDs was analyzed using Fourier transform infrared spectroscopy  in the range 400–4000 cm2SO4 were prepared. The absorbance of both wh-CQDs and quinine sulfate solution was adjusted to below 0.1 to reduce the internal filtration effect. The QY of wh-CQDs was calculated using the following Equation (1)  is the Fe3+ concentration. The detection limit was calculated using the formula LOD = 3σ/S, where σ represents the standard deviation of the fluorescence intensity of the blank wh-CQDs, and S is the slope of the linear function (n = 10). From the results obtained, we calculated the detection limit to be 0.084 μM. Notably, the obtained detection limit of Fe3+ by the wh-CQDs was much lower than that proposed by the World Health Organization (5.36 μM), indicating that the wh-CQDs can be applied in highly selective sensing of Fe3+. A comparison of different systems for Fe3+ determination is summarized in For the sensitivity experiments, the fluorescence intensities at different concentrations of Feon light b. The flo 330 μM c. The raKlebsiella sp. was used as a model for toxicity studies. As shown in Klebsiella sp.CQDs are used as probes in various fields owing to their excellent tunable fluorescence properties, colorful fluorescence emission, small size, high photostability, and biocompatibility ,65,66. IKlebsiella sp. in a medium containing 500 μg/mL wh-CQDs for 12 h. As observed using laser confocal microscopy, the CQDs exhibited an intense blue fluorescence signal at excitation wavelengths of 375–407 nm with outstanding imaging potential. Notably, Klebsiella sp. cell walls contain various polysaccharides and lipids, which make them difficult to be labeled. The wh-CQDs could enter the bacterial membrane via endocytosis, indicating that the wh-CQDs have strong penetrating ability. Therefore, owing to the strong fluorescence, stability, and good biocompatibility of wh-CQDs, they are expected to be an effective fluorescent probe for bacterial cell tracers in the bioenergy field.As shown in Klebsiella sp. cells. To this end, cells were incubated with 500 μg/mL wh-CQDs and analyzed by flow cytometry after incubation for 12 and 24 h at 37 °C. The emission of wh-CQDs was analyzed in the FITC fluorescence channel. As shown in 5 CFU/mL of unstained Klebsiella sp. was extremely weak, and the fluorescence was mainly biological autofluorescence. As shown in Klebsiella sp. labeled with wh-CQDs in the P2 region was significantly enhanced, which could be distinguished from the unstained cell clusters. The P2 region incubated for 12 h accounted for 32.89% of the total cell number, indicating that the proportion of wh-CQDs-labeled Klebsiella sp. accounted for 32.89% of the total cell number. Similarly, in Klebsiella sp. cells.Based on the cellular imaging results, we determined the proportion of wh-CQD uptake by 3+. In addition, toxicity and imaging studies on the hydrogen-producing bacterium Klebsiella sp. and flow cytometry analysis of intracellular wh-CQDs were performed for the first time using biomass-derived carbon dots. Owing to the green synthesis of wh-CQDs, even at high concentrations (1000 μg/mL), wh-CQDs showed low cytotoxicity toward hydrogen-producing bacteria. Notably, the synthesized wh-CQDs possessed strong penetrating ability and good biocompatibility, and were able to break through the lipid and polysaccharide barrier in Klebsiella sp. cell wall. The wh-CQDs entered bacterial cells and emitted bright blue fluorescence, implying that they may be an effective probe for flow cytometry. However, the long-term performance of wh-CQDs tracer analysis in Klebsiella sp. is still lacking, and the analysis of bacterial cell imaging mechanism is not deep enough, which needs to be further improved in the future research work. Overall, our study expands the application of biomass carbon spots and demonstrates that wh carbon spots can be used as sensitive fluorescent probes for Fe3+ detection and cellular imaging.In the present study, blue fluorescent CQDs were derived from natural biomass wh using a bottom-up hydrothermal method at 180 °C for 12 h. The synthesized wh-CQDs were uniform in size, with an average size of 2.44 ± 0.57 nm, and exhibited typical excitation-light-dependent properties owing to surface state defects. The wh-CQDs exhibited excellent physicochemical properties with high selectivity and sensitivity to Fe


I–V characteristics with a lower current density leakage than those with the p-ZnO/GZO LED structure. Moreover, the emission intensity was improved by adding the ZnMgO NP layer to the LEDs. These results show that the ZnMgO NP layer acts as a hetero-barrier layer that suppresses the diffusion of holes into the n-type layer and confines holes to the p-type layer.Heterojunction light-emitting diodes (LEDs), based on p-type ZnO and n-type ZnMgO nanoparticles, have been demonstrated. ZnMgO nanoparticles were prepared by the thermal diffusion of Mg onto ZnO nanoparticles. p-ZnO/GZO homostructure LEDs and p-ZnO/n-ZnMgO/GZO heterostructure LEDs have been fabricated using ZnO and ZnMgO nanoparticles. By comparing the characteristic results of these diodes, it can be seen that LEDs with the p-ZnO/n-ZnMgO/GZO structure showed better  Light-emitting diodes (LEDs) have vast potential in the development of solid-state lighting technologies due to their prominent properties. Currently, the fabrication of light-emitting diodes based on ZnO is of great interest because of their advantageous features, such as a wide bandgap of 3.37 eV, excitonic binding energy of 60 meV, and short wavelength UV emission range at room temperature ,2. To acRecently, ZnO NP has been considered a promising material for the development of semiconductor devices. Some groups have proposed developing nitrogen-doped ZnO NPs using different techniques ,12,13. OIt was realized that, to overcome this problem, these devices can be constructed based on a heterostructure because carrier confinement is an essential requirement for improving luminescence properties and can lead to high radiative recombination in the active region of the devices. One of the necessary processes of device construction is bandgap engineering by changing the energy structure. The energy structure of the materials has been changed by doping, surface modification, and controlling the size ,21,22. S2 radicals generated by the arc plasma, together with nitrogen atoms, were incorporated into ZnO NPs during the synthesis process. In this method, we used zinc metal  as a Zn source and dry air as an oxygen and nitrogen source. During the synthesis process, the evaporated zinc atoms were reacted with flowing gas, and ZnO NPs were produced. The chamber pressure was regulated at 150 torr and 610 torr through a rotary pump and controlling valve. The arc currents were 30 A and 50 A. The airflow rate inside the chamber was 5 L min−1 during the synthesis period. The detailed mechanism of the synthesis of ZnO NPs was described in ta previous report [A gas evaporation method  was applied to synthesize the N-doped ZnO NPs by an arc plasma system and an oxidation system. The Ns report ,15. The s report ,30. The s report ,19. 2 gas flow of 0.5 L min−1 inside the furnace. The furnace temperature was increased from room temperature to 1000 °C within 6 min and kept constant for 60 min. The NP samples were cooled down to less than 40 °C in 2 h.For the preparation of ZnMgO composite NPs, the prepared n-ZnO NP samples were fabricated at a pressure of 610 torr and an arc current of 50 A, which exhibited n-type conductivity . The n-Z−4 Ω cm). The n-ZnMgO-composite NP dispersions were prepared by mixing 10 g pure water with 0.1 g ZnMgO NPs using an ultrasonic homogenizer. Large-size particles were separated from the dispersion by a centrifugal separator  . For the formation of the n-ZnMgO NP layer, the separated dispersion was sprayed using an airbrush process onto the GZO electrode film, which was on the hotplate at a temperature of ~300 °C [Two types of ZnO-NP-based LED were designed following the methods earlier ,19. One For the fabrication of the p-type ZnO NP layer in the LEDs, the dispersion was prepared by mixing with N-doped ZnO NPs (0.05 g), isopropyl alcohol (IPA) (0.3) mL, and binder (0.1 g) . The p-ZnO NP dispersions were coated on the GZO electrode films by the spin-coating process. The spin-coating process followed a two-step rotational condition at an initial speed of 1000 rpm for 5 s and was accelerated to a final speed of 4000 rpm for 10 s. The nitrogen-doped ZnO NP-coated layers were sintered by a hotplate at ~300 °C. Gold (Au) electrodes with 30 nm thickness were deposited on both the p-type layer and the GZO film, as a contact electrode, using a vacuum deposition procedure. The thickness of the spin-coated layer was approximately 3 µm and the thickness of the spray-coated layer was around 2 µm according to a previous report ,19. α radiation was used to measure X-ray diffraction of the NP powder samples. A Horiba FluoroMax-4 spectrofluorometer with an excitation wavelength of 325 nm, using a Xe lamp, was used to detect the photoluminescence (PL) spectra of the ZnO NPs. The current–voltage measurements of the light-emitting diodes were performed using a parameter analyzer (Keysight Technologies B2900A series of High-Resolution SMU module). We evaluated the electroluminescence (EL) spectra of the LEDs from the top side of the p-contact electrode at room temperature using the Ocean Optics QE65000 fiber multi-channel monochrome meter.A diffractometer (Rigaku Smart Lab) with Cuk+2) imposed in the host ZnO lattice. This attribution was due to the difference in ionic radius, where the ionic radius of Mg is 0.57 Å, and that of Zn is 0.60 Å [1−xMgxO NP composite, the equation isx) in the composite NPs was calculated to be 0.18 (Zn0.82Mg0.18O).s 0.60 Å ,32,33. Ts 0.60 Å . Vegard’s 0.60 Å . For thes 3.32 Å ,37. The x) of Mg can be calculated from Equation (2) [tion (2) .Eg(ZnMgx) of Mg in the composite ZnMgO NPs was determined to be 0.01. This is different from the results of the XRD. From the XRD results, the content of Mg (x) was 0.18. In this case, the bandgap of ZnMgO was 3.956 eV. The reason for this difference is the lack of uniformity in the thermal diffusion process. The large amount of Mg diffused onto the surface of the ZnO NPs and this distortion was revealed by the XRD. However, the PL emission was less affected because of the many defects on the surface of the NPs. Assuming that the bandgap of MgO is 6.626 eV ,39, the c, Ev, and EF are, respectively, the conduction band, valence band and quasi-Fermi level energy. We considered the working mechanism of the ZnO NP-based homo- and heterostructure LEDs. I–V properties of these LEDs were evaluated. I–V characteristics of LEDs at room temperature in a dark environment. Nonlinear rectifying behavior was observed from the I–V curve for both structures of LED. The p-ZnO/n-ZnMgO/GZO LED showed superior characteristics compared to the p-ZnO/GZO LED. The turn-on voltage for the LEDs with the structure of p-ZnO/n-ZnMgO/GZO was at around 3.3 V at the forward region, which is in good agreement with the bandgap energy of ZnO. However, this was not observed for the LEDs with the structure of p-ZnO/GZO, which also revealed a higher current density than the LEDs with the structure of p-ZnO/n-ZnMgO/GZO; this is possibly due to the interface difference between the p-ZnO NP and the n-GZO film layers and the interface of the p-ZnO/n-ZnMgO NP layer. On the other hand, the active region for the both structures of LED exists in the p-ZnO NP layer that contains an insulating binder and also the n-type NPs, due to the non-homogeneous particle fabrication process.The The EL spectra of the LEDs with p-ZnO/n-ZnMgO/GZO and p-ZnO/GZO structures at room temperature are presented in Eg (T = 0) = 3.44 eV for ZnO at 0 K [α = −5.5 × 10−4 eV K−1 and β = −900 K for temperatures up to 300 K [The peak energy of EL emission for p-ZnO/GZO LEDs was found to be around 3.23 eV. On the other hand, the peak energy of EL spectra for the LED with an inserted ZnMgO NP layer (p-ZnO/n-ZnMgO/GZO) was observed at about 3.21 eV. When comparing the EL spectra for both LEDs, the emission energy peaks were shifted toward the lower-energy side. The results prove that the EL emission occurred by carrier recombination in the p-ZnO NP layer. The lower direction of the peak energy shift is due to the decreased bandgap energy, caused by the heating effect of the rising temperature , and theO at 0 K . The temto 300 K ,43. The As mentioned above, the Mg content of the ZnMgO NPs influenced the ZnO structure due to bandgap tuning. Considering these results, the authors conclude that the ZnMgO NP layer acted as an n-type layer in the diode and created a hetero-barrier for hole injection in the active region of the LEDs.1−xMgxO could be optimized for significant progress in optoelectronic devices.By compositing Mg into ZnO NPs, the bandgap engineering of ZnMgO NPs was confirmed and the characterization was revealed by the results, including XRD and PL. The ZnMgO NP layer was inserted into the LEDs as an energy barrier layer, which created carrier confinement in the active region of the devices and carried out electron–hole balance radiative recombination. The diode characteristics of the LEDs with the structure of p-ZnO/n-ZnMgO/GZO were significantly improved, as well as the EL emission, which was comparable to that of the p-ZnO/GZO LED structure. In light of these results, the structure and fabrication processes of heterostructure LEDs with Zn